Bacteria can become resistant to antibiotics via three main mechanisms: antibiotic inactivation, target modification and alteration of intracellular antibiotic concentration. The latter mechanism can occur by either decreasing permeability to an antibiotic or by increasing the activities of a variety of efflux pumps. While permeability is a significant barrier to antibiotics in gram-negative bacteria, due to the presence of an outer membrane, it is an unlikely mechanism of resistance for gram-positive bacteria that lack an outer membrane. Both gram-positive and gram-negative bacteria possess multiple, chromosomal- and plasmid-encoded efflux pumps with broad substrate specificities. Putnam et al., “Molecular properties of bacterial multidrug transporters,” Microbiol. and Molecular Biol. Rev., 64:672-693 (2000); Munoz-Bellido et al., “Efflux-mediated antibiotic resistance in Gram positive bacteria,” Reviews. Med. Microbiol., 13:1-13 (2002); Bambeke et al., “Antibiotic efflux pumps,” Biochem. Pharmacol., 60:457-470 (2000).
One natural role of efflux pumps in prokaryotic and eukaryotic cells is to remove toxins from the interior of the cell. This protective function enables bacterial cells to survive in hostile environments, including the presence of antibiotics during the treatment of infections. Efflux of antibiotics is a clinically significant general resistance mechanism for bacteria. Kohler et al., “Bacterial antibiotic efflux systems of medical importance,” Cell. Mol. Life Sci., 56:771-778 (1999). The up-regulation of efflux systems through physiological induction and spontaneous mutation can significantly lower the intracellular concentration of many antibiotics, causing an impact on clinical efficacy. Bacteria can express multiple efflux pumps which are capable of extruding a wide variety of structurally unrelated compounds, including both naturally and synthetically produced antibiotics. For instance, the sequence of the S. aureus genome indicates that this organism may possess up to 17 drug transporters or more since an analysis of the genome sequence of methicillin-resistant Staphylococcus aureus N315 indicates that there are >20 open reading frames capable of encoding antibiotic efflux pumps. Kuroda et al., “Whole genome sequencing of methicillin-resistant Staphylococcus aureus,” Lancet, 357:1225-1240 (2001) and http://www.membranetransport.org.
For gram-negative bacteria, the resistance-nodulation-cell division (RND) family of pumps play the greatest role in contributing to resistance to clinically relevant antibiotics. Examples of this class of efflux pumps include the AcrB pump in E. coli and the MexB, D, F and Y pumps in P. aeruginosa (Bambeke et al., “Antibiotic efflux pumps”, Biochem. Pharmacol. 60:457-470 (2000) and Putman, van Veen and Konings, “Molecular properties of bacterial multidrug transporters”, Microbiol. Mol. Biol. Rev. 64:672-693 (2000)).
To date, RND pumps have not been described in gram-positive organisms. For gram-positive bacteria, the major facilitator superfamily class (MFS) pumps play the greater role in the efflux of clinically relevant antibiotics, contributing to clinical resistance. MFS pumps have been found in both prokaryotes and eukaryotes, including mammals, and examples of this class of efflux pumps include the NorA pump in S. aureus (Neyfakh et al., “Fluoroquinolone resistance protein NorA of Staphylococcus aureus is a multidrug efflux transporter”, Antimicrob. Agents Chemother. 37:128-129 (1993)), the PmrA pump in S. pneumoniae (Gill et al., “Identification of an efflux pump gene, pmrA, associated with fluoroquinolone resistance in Streptococcus pneumoniae,” Antimicrob. Agents Chemother., 43:187-189 (1999)), and the EmeA pump of E. faecalis (Lee et al., “Functional cloning and expression of emeA, and characterization of EmeA, a multidrug efflux pump from Enterococcus faecalis,” Biol. Pharm. Bull., 26:266-270 (2003)).
Recent reports have described the crystal structures of the E. coli AcrB pump (Murakami et al., “Crystal structure of bacterial multidrug efflux transporter AcrB”, Nature 419:587-593 (2002) and Yu et al., “Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pump”, Science 300:976-980 (2003)) and the Bacillus subtilis BmrR MDR transcriptional activator (Zheleznova et al., “Structural basis of multidrug recognition by BmrR, a transcription activator of a multidrug transporter”, Cell 96:353-362 (1999)), both co-complexed with substrates, and the outer membrane transporter TolC (Koronakis et al., “Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export”, Nature 405:914-919 (2000)). While structural details provide the basis for substrate recognition, the mechanism by which molecules are actually transported to the outside of a cell remains to be elucidated. In addition, while there is some degree of substrate overlap between RND- and MFS-type pumps, there are sufficient differences in their substrate specificities and structures to explain that several known pump inhibitors only inhibit one family or the other, but not both.
Inhibition of efflux is one way to increase the clinical efficacy of an antibiotic even in the presence of target-based mutations. In response to emerging resistance to all classes of antibiotics, in particular fluoroquinolones, this has been a significant focus of the pharmaceutical industry (Lawrence and Barrett, “Inhibition of bacterial efflux: needs, opportunities, and strategies,” Curr. Opin. Antiinfect. Invest. Drugs, 2:145-153 (2000)). Many pharmaceutical industry programs have focused on identifying inhibitors of gram-negative and gram-positive efflux systems that could potentially be used in combination with antibiotics to improve their efficacy and suppress resistance (Aeschlimann et al., “Effects of NorA inhibitors on in vitro antibacterial activities and postantibioitic effects of levofloxacin, ciprofloxacin, and norfloxacin in genetically related strains of Staphylococcus aureus”, Antimicrob. Agents Chemother. 43:335-340 (1999); Bambeke et al., “Antibiotic efflux pumps”, Biochem. Pharmacol. 60:457-470 (2000); Germann et al., “Cellular and biochemical characterization of VX-710 as a chemosensitizer: reversal of P-glyco-protein-mediated multidrug resistance in vitro”, Anticancer Drugs 8:125-140 (1997); Kaatz et al., “Identification and characterization of a novel efflux-related multidrug resistance phenotype in Staphylococcus aureus”, J. Antimicrob. Chemother. 50:833-838 (2002); Kuroda et al., “Whole genome sequencing of methicillin-resistant Staphylococcus aureus”, Lancet 357:1225-1240 (2001); Lee et al., “Functional cloning and expression of emeA, and characterization of EmeA, a multidrug efflux pump from Enterococcus faecalis”, Biol. Pharm. Bull. 26:266-270 (2003); Markham et al., “Inhibition of the multidrug transporter NorA prevents emergence of norfloxacin resistance in Staphylococcus aureus”, Antimicrob. Agents Chemother. 40:2673-2674 (1996); Putman et al., “Molecular properties of bacterial multidrug transporters”, Microbiol. Mol. Biol. Rev. 64:672-693 (2000); Renau et al., “Conformationally-restricted analogues of efflux pump inhibitors that potentiate the activity of levofloxacin in Pseudomonas aeruginosa”, Bioorg. Med. Chem. Lett. 13:2755-2758 (2003); Rowinsky et al., “Phase I and pharmacokinetic study of paclitaxel in combination with biricodar, a novel agent that reverses multidrug resistance conferred by overexpression of both MDR1 and MRP”, J. Clin. Oncol. 16:2964-2976 (1998)).
In vitro, efflux pump inhibitors (EPIs) have been shown to reduce spontaneous resistance frequencies of antibiotics in P. aeruginosa (Lomovskaya et al., “Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy”, Antimicrob. Agents Chemother. 45:105-116 (2001)), S. pneumoniae (Markham et al., “Inhibition of the emergence of ciprofloxacin resistance in Streptococcus pneumoniae by the multidrug efflux inhibitor reserpine”, Antimicrob. Agents Chemother. 43:988-989 (1999)), and S. aureus (Markham et al., “Inhibition of the multidrug transporter NorA prevents emergence of norfloxacin resistance in Staphylococcus aureus”, Antimicrob. Agents Chemother. 40:2673-2674 (1996); Markham et al., “Multiple Novel Inhibitors of the NorA Multidrug Transporter of Staphylococcus aureus”, Antimicrob. Agents Chemother. 43:2404-2408 (1999)). In an animal model of P. aeruginosa infection, Renau, et al., “Inhibitors of efflux pumps in Pseudomonas aeruginosa potentiate the activity of the fluoroquinolone levofloxacin”, J. Med. Chem. 42:4928-4931 (1999)) showed that levofloxacin plus an EPI was more efficacious than levofloxacin alone, demonstrating the potential for combination therapy in vivo.
Reserpine, a plant alkaloid, is a known inhibitor of both mammalian and gram-positive bacterial efflux whose clinical utility is limited by neurotoxicity (Neyfakh, et al., “Efflux-mediated multidrug resistance in Bacillus subtilis: Similarities and dissimilarities with the mammalian system,” Proc. Nat'l Acad. Sci., 88:4781-4785 (1991)). Reserpine has activity against the MFS S. aureus NorA pump, a well-known contributor to fluoroquinolone resistance in this organism. Homologs of the NorA pump can be found in multiple gram-positive bacteria suggesting that reserpine, and other NorA pump inhibitors, would work with other clinical pathogens.
Thus, there is a need for compounds that potentiate the activity of an antibacterial (e.g., an antibiotic). There is also a need for compositions useful in treating bacterial infection in mammals, and methods therewith. There is also a need for a method of inhibiting bacterial efflux of an antibiotic, thereby increasing the efficacy of the antibiotic.